Search

Warfarin, a widely prescribed anticoagulant, is highly effective for various coagulation disorders. However, its efficacy is limited by a narrow therapeutic index and frequent drug interactions, especially those involving metabolism by Cytochrome P450 (CYP450) enzymes. Piperine, found in black and long pepper, possesses blood-thinning properties and has been observed to inhibit CYP3A and CYP2C enzymes linked to warfarin metabolism. This study investigated the effect of piperine on warfarin metabolism in liver microsomes using a rapid and sensitive HPLC-Fluorescence method. The use of PFP (pentafluorophenyl) column with core shell particles provided the selectivity and resolution to resolve warfarin and its 4-, 6-, 7-, and 10-hydroxy metabolites in addition to the internal standard naproxen in less than 3 min. This is the fastest analytical assay for warfarin and its major metabolites reported to date, making it ideal for metabolic studies. The applicability of the method was demonstrated by monitoring the metabolism of S-warfarin in human and rat liver microsomes, and evaluating the inhibitory effect of piperine on metabolite formation. The results showed that piperine inhibited the formation of the major metabolite, 7-hydroxywarfarin, with half-maximal inhibitory concentration (IC50) 14.2 μM and 3.2 μM in human and rat liver microsomes, respectively. Furthermore, coagulation studies in vitro using rat plasma showed that piperine does not affect prothrombin time (PT) and activated partial thromboplastin time (aPTT). This study suggested that piperine may present a potential drug interaction with warfarin at the metabolism level, but has no direct effect on the activation of the extrinsic or intrinsic coagulation cascades. Further clinical investigation is therefore required, as piperine may increase the bioavailability of warfarin, thus increasing risk of serious adverse events in patients.

In this study, high-performance liquid chromatography with fluorescence detection (HPLC-FLD) has been used for the first time, for direct determination of warfarin and its major metabolite, 7-hydroxywarfarin, in rat plasma. The simple and sensitive method was developed using Fortis® reversed-phase diphenyl column (150 × 4.6 mm, 3 μm) and a mobile phase composed of phosphate buffer (25 mmol L−1)/methanol/acetonitrile (70:20:10, V/V/V), adjusted to pH 7.4, at a flow rate of 0.8 mL min−1. The diphenyl chemistry of the stationary phase provided a unique selectivity for separating the structurally related aromatic analytes, warfarin and 7-hydroxywarfarin, allowing their successful quantification in the complex plasma matrix. The method was linear over the range 0.01–25 μg mL−1, for warfarin and 7-hydroxywarfarin, and was found to be accurate, precise and selective in accordance with US FDA guidance for bioanalytical method validation. The method was sensitive enough to quantify 0.01 μg mL−1 of warfarin and 7-hydroxywarfarin (LLOQ) using only 100 μL of plasma. The applicability of this method was demonstrated by analyzing samples obtained from rats after oral administration of a single warfarin dose, and studying warfarin and 7-hydroxywarfarin pharmacokinetics.

Aref Zayed,1 Wahby M Babaresh,1 Ruba S Darweesh,2 Tamam El-Elimat,1 Sahar S Hawamdeh1 1Department of Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy, Jordan University of Science and Technology, Irbid 22110, Jordan; 2Department of Pharmaceutical Technology, Faculty of Pharmacy, Jordan University of Science and Technology, Irbid 22110, JordanCorrespondence: Aref Zayed Tel +962 2 7201000, Ext. 23240Fax +962 2 7201075Email alzayed@just.edu.joIntroduction: Piperine, the bioactive compound of black pepper, and warfarin are metabolized by cytochrome P450 enzymes and are both highly plasma protein-bound compounds. In this study, we evaluated the effect of co-administered piperine on the pharmacokinetics and anticoagulation of warfarin in rats.Methods: We studied four Sprague-Dawley rat groups: a negative control group receiving only oral warfarin, a test group receiving warfarin plus piperine, a positive control group receiving warfarin plus sulfaphenazole (CYP2C inhibitor), and another positive control group receiving warfarin plus ketoconazole (CYP3A inhibitor). We also analyzed plasma concentrations of warfarin and its major metabolite, 7-hydoxywarfarin. Blood clotting time, calculated as international normalized ratio (INR), was also measured.Results: Our results showed that although co-administration of piperine produced a non-significant decrease in warfarin concentrations, it resulted in significantly lower 7-hydroxywarfarin metabolite concentrations. Piperine significantly decreased, by sixfold, AUC0–∞, by eightfold, Cmax, but significantly increased, by fivefold, CL/F and, by sixfold, Vd/F of 7-hydroxywarfarin. The INR values were consistent with the decrease in warfarin concentration in the presence of piperine and showed a significant decrease at 24 h after warfarin dose.Conclusion: We conclude that piperine could be a potent inhibitor of cytochrome P450 metabolism of warfarin in vivo and, contrary to the expectation, may reduce the plasma concentration and anticoagulation of warfarin. This interaction could have a clinical significance and should be investigated in patients.Keywords: warfarin, 7-hydroxywarfarin, piperine, pharmacokinetics, herb–drug interaction, black pepper

The widely used anticoagulant Coumadin ( R / S -warfarin) undergoes oxidation by cytochromes P450 into hydroxywarfarins that subsequently become conjugated for excretion in urine. Hydroxywarfarins may modulate warfarin metabolism transcriptionally or through direct inhibition of cytochromes P450 and thus, UGT action toward hydroxywarfarin elimination may impact levels of the parent drugs and patient responses. Nevertheless, relatively little is known about conjugation by UDP-glucuronosyltransferases in warfarin metabolism. Herein, we identified probable conjugation sites, kinetic mechanisms and hepatic UGT isoforms involved in microsomal glucuronidation of R - and S -7-hydroxywarfarin. Both compounds underwent glucuronidation at C4 and C7 hydroxyl groups based on elution properties and spectral characteristics. Their formation demonstrated regio- and enantioselectivity by UGTs and resulted in either Michaelis–Menten or substrate inhibition kinetics. Glucuronidation at the C7 hydroxyl group occurred more readily than at the C4 group, and the reaction was overall more efficient for R -7-hydroxywarfarin due to higher affinity and rates of turnover. The use of these mechanisms and parameters to model in vivo clearance demonstrated that contributions of substrate inhibition would lead to underestimation of metabolic clearance than that predicted by Michaelis–Menten kinetics. Lastly, these processes were driven by multiple UGTs indicating redundancy in glucuronidation pathways and ultimately metabolic clearance of R - and S -7-hydroxywarfarin. [ABSTRACT FROM AUTHOR]

[Display omitted] • Electrooxidation of (S)-7-hydroxywarfarin was investigated. • Electroanalytical characteristics of (S)-7-hydroxywarfarin were determined. • Electrooxidation of (S)-7-hydroxywarfarin was used for assessment of CYP2C9 activity. An electrochemical method for the quantitative determination of (S)-7-hydroxywarfarin as a metabolite of the vitamin K antagonist warfarin has been developed to analyze the catalytic activity of cytochrome P450 2C9 (CYP2C9). The electrochemical properties of (S)-7-hydroxywarfarin were investigated using screen-printed carbon electrodes. We have shown by cyclic voltammetry that irreversible (S)-7-hydroxywarfarin electrochemical oxidation, in contrast to (S)-warfarin, can be registered by an oxidation peak at approximately 0.6 V (vs. Ag pseudo-reference electrode). A linear dependence of the oxidation peak amplitude in the range of 0.57–0.61 V on (S)-7-hydroxywarfarin concentration (in the range of 0.1–1 μM) was shown by the square-wave voltammetry method. The limit of (S)-7-hydroxywarfarin detection and the sensitivity were calculated as 0.091 μM and 0.0075 A/M, respectively. Using an electrochemical system based on recombinant human CYP2C9 immobilized on screen-printed carbon electrodes modified by didodecyldimethylammonium bromide, we have shown the possibility of (S)-7-hydroxywarfarin electrochemical quantification without separation of the enzymatic system component. The (S)-7-hydroxywarfarin quantitative electrochemical determination was used to calculate the kinetic parameters of CYP2C9 immobilized on electrode toward (S)-warfarin: the values of the maximal reaction rate (V max) and the Michaelis constant (K M) were calculated as 0.1 ± 0.002 min−1 and 3.03 ± 0.38 μM, respectively. The usage of an enzymatic CYP2C9-containing electrode as a catalyst and an indicator electrode as a sensor for the formation of metabolite allows to effectively register the kinetic parameters of the process. Such a two-electrode system can be applied to assess drug-drug interactions. [ABSTRACT FROM AUTHOR]